Function
PAPER
Cite this: Food Funct., 2016, 7, 1458
Received 11th December 2015, Accepted 2nd February 2016 DOI: 10.1039/c5fo01536g www.rsc.org/foodfunction
Bioactivity, proximate, mineral and volatile pro
files
along the
flowering stages of Opuntia microdasys
(Lehm.): de
fining potential applications
Hassiba Chahdoura,
a,b,cJoão C. M. Barreira,*
aVirginia Fernández-Ruiz,
dPatricia Morales,
dRicardo C. Calhelha,
aGuido Flamini,
e,fMarina Sokovi
ć,
gIsabel C. F. R. Ferreira*
aand Lot
fi Achour
bOpuntia spp.flowers have been traditionally used for medical purposes, mostly because of their diversity in bioactive molecules with health promoting properties. The proximate, mineral and volatile compound profiles, together with the cytotoxic and antimicrobial properties were characterized in O. microdasys flowers at different maturity stages, revealing several statistically significant differences. O. microdasys stood out mainly for its high contents of dietary fiber, potassium and camphor, and its high activities against HCT15 cells, Staphylococcus aureus, Aspergillus versicolor and Penicillium funiculosum. The vegetative stage showed the highest cytotoxic and antifungal activities, whilst the fullflowering stage was particularly active against bacterial species. The complete dataset has been classified by principal com-ponent analysis, achieving clearly identifiable groups for each flowering stage, elucidating also the most distinctive features, and comprehensively profiling each of the assayed stages. The results might be useful to define the best flowering stage considering practical application purposes.
Introduction
Among angiosperms, the Cactaceae is one of the most distinc-tive and successful families of plants of the New World, com-prising more than 1600 species.1Plants in the genus Opuntia are members of the Cactaceae family, being widely distributed in semi-arid countries throughout the world, especially in the Mediterranean area and Central America.2,3 Opuntia spp. flowers have been traditionally used for medical purposes for a long time. The dried flowers of prickly pear are usually sold in the popular Tunisian markets, being commonly used as
infu-sions in traditional Tunisian and in Sicilian medicine for their diuretic activity, capacity to help in renal calculus expulsion and to cure ulcer.4,5In fact, different parts of Opuntia sp. are being increasingly used in nutritional and pharmacological applications (including at the industrial level). Nevertheless, the number of reports characterizing the chemical profiles of their flowers is still scarce, especially throughout their ripen-ing, which motivated our present investigation.
The methanolic extracts from flowers of Opuntia microdasys (Lehm.) have been recently reported as containing high quan-tities of polyphenols (especially flavonoids) and a strong antioxi-dant activity.6 The interest in plant materials containing phenolic compounds is increasing due to their high antioxidant potency, which may offer protection against different diseases, such as cancer through the inhibition of oxidative damage (known for being a potential cause of mutation).7Furthermore, there is a rising awareness regarding the use of volatiles in both the food and the pharmaceutical industries, justifying a sys-tematic examination of plant extracts for these compounds.8
Despite only a few reports describe the volatile composition of Opuntia spp. flowers, it is possible to point out tetra-decanoic acid, hexatetra-decanoic acid, octadecadienoic acid and camphor as the main volatile compounds.9–11Opuntia flowers have also been reported to contain high levels of minerals (e.g., K, Ca, Mg) and fiber.12 Likewise, there are some pub-lished studies describing their antibacterial activity.11,12
aMountain Research Centre (CIMO), ESA, Polytechnic Institute of Bragança, Campus de Santa Apolónia, Ap. 1172, 5301-855 Bragança, Portugal. E-mail: jbarreira@ipb.pt, iferreira@ipb.pt; Fax: +351273325405; Tel: +351273303903, +351273303219 bLaboratoire de Recherche“Bioressources”: Biologie Intégrative & Valorisation, Institut Supérieur de Biotechnologie de Monastir, Avenue Tahar Hadded, BP 74, 5000, Université de Monastir, Monastir, Tunisia
cDépartement des Sciences de la Vie. Faculté des Sciences de Bizerte, Université de Carthage, Tunisia
dDpto. Nutrición y Bromatología II, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), Plaza Ramón y Cajal, s/n. E-28040 Madrid, Spain
eDipartimento di Farmacia, via Bonanno 6, 56126 Pisa, Italy
fCentro Interdipartimentale di Ricerca“Nutraceutica e Alimentazione per la Salute” Nutrafood, University of Pisa, Italy
gDepartment of Plant Physiology, Institute for Biological Research“Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia
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However, the effect of the phenological stage in which the components are evaluated was not studied for this species. This is an important research topic, since the amounts and nature of compounds may vary along the flowering stage, suggesting changes in the secondary metabolism of flowers.
Overall, this study seeks to contribute to the knowledge of the nutritional value and biological properties of O. microdasys flowers, characterizing their potential use as a functional food. Furthermore, the flowers’ volatile composition at different flow-ering stages has never been reported before in this species.
Experimental
Samples
Opuntia microdasys (Lehm.) flowers were collected in 2013 from the cliff of Monastir (Tunisia) at three phenological stages: (F1) the vegetative stage, with green closed petal flowers (harvested in the beginning of June); (F2) the full flowering stage: stamens are separated around the style, the flowers are fully opened and the nectar production starts (harvested after the first fortnight of June); (F3) the post flowering stage: the flowers are dried and close to senescence (obtained in the last week of June). Samples for analysis (50 g for each flowering stage) were dried in the shade, ground using a Warring blender (Phillips, France), reduced to a fine dried powder (20 mesh) and mixed to obtain a homogeneous sample. Standards and reagents
Micro (Fe, Cu, Mn and Zn) and macroelement (Ca, Mg, Na and K) standards (>99% purity), as well as LaCl2 and CsCl (>99% purity) were purchased from Merck (Darmstadt, Germany). Mueller–Hinton agar (MH) and malt agar (MA) were obtained from the Institute of Immunology and Virology, Torlak (Bel-grade, Serbia). Acetic acid, ellipticine, phosphate buffered saline (PBS), acetic acid, sulforhodamine B (SRB), trichloroace-tic acid (TCA), Tris, streptomycin (Sigma-Aldrich S6501) and ampicillin (Sigma-Aldrich A9393) were purchased from Sigma (St Louis, MO, USA). Bifonazole (Srbolek, Belgrade, Serbia) and ketoconazole (Zorkapharma, Šabac, Serbia) were used as the reference fungicides.
Foetal bovine serum (FBS), L-glutamine, Hank’s balanced
salt solution (HBSS), trypsin-EDTA (ethylenediamine tetra-acetic acid), nonessential amino acid solution (2 mM), penicil-lin/streptomycin solution (100 U mL−1 and 100 mg mL−1, respectively), RPMI-1640 and DMEM media were from Hyclone (Logan, UT, USA). Dimethylsulfoxide (DMSO), (Merck KGaA, Darmstadt, Germany) was used as a solvent. Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA). All other chemicals and solvents were of analytical grade and purchased from common sources. Proximate analysis and fiber composition
The samples were analyzed for chemical composition (moist-ure, proteins, fat and carbohydrates) using the AOAC pro-cedures.13 The crude protein content (AOAC 928.08) of the
samples was estimated by the macro-Kjeldahl method (N × 6.25); the crude fat (AOAC 991.36) was determined by extract-ing a known weight of the powdered sample with petroleum ether in a Soxhlet apparatus; the ash content was determined by incineration at 550 ± 15 °C. Total carbohydrates were calcu-lated by subtracting the amounts of protein, ash and fat, con-sidering 100 g of dried sample.
Soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) were determined according to the AOAC enzymatic–gravimetric method (993.19 and 991.42).14 Freeze-dried samples were treated with alpha-amylase (heat-stable), protease and amylo-glucosidase. The soluble and insoluble fractions were separ-ated by vacuum filtration. Waste from the digests was dried at 100 °C. Total dietary fiber (TDF) was the sum of SDF and IDF; both were expressed as g per 100 g of dry weight.
The energy value was calculated according to the following equation: Energy (kcal per 100 g dw) = 4 × (g protein) + 4 × (g carbohydrate− g TDF) + 2 × (TDF) + 9 × (g fat).
Ash content and mineral composition
The method 930.05 of AOAC was used. A sample of 500 mg was incinerated under high pressure in a microwave oven (Muffle Furnace mLs1200, Thermo Scientific, Madrid, Spain) for 24 h at 550 °C, and ashes were gravimetrically quantified. The residue of incineration was extracted with HCl (50%, v/v) and HNO3(50%, v/v) and made up to an appropriate volume with distilled water, where Fe, Cu, Mn and Zn were directly measured. An additional 1/10 (v/v) dilution of the sample extracts and standards was performed to avoid interferences between different elements in the atomic absorption spec-troscopy: for Ca and Mg analysis in 1.16% La2O3/HCl (leading to LaCl2); and for Na and K analysis in 0.2% CsCl.15 All measurements were performed using atomic absorption spec-troscopy (AAS) by using Analyst 200 Perkin Elmer equipment (Perkin Elmer, Waltham, MA, USA), comparing absorbance responses with 99.9% purity analytical standard solutions for AAS made with Fe(NO3)3, Cu(NO3)2, Mn(NO3)2, Zn(NO3)2, NaCl, KCl, CaCO3and the Mg band, supplied by Merck (Darm-stadt, Germany) and Panreac Química (Barcelona, Spain). Volatile compound analyses
Supelco (Bellefonte, PA) SPME devices coated with poly-di-methylsiloxane (PDMS, 100μm) were used to sample the head-space of a dry flower inserted into a 5 mL vial and allowed to equilibrate for 30 min. SPME sampling was performed using the same new fiber, preconditioned according to the manufac-turer’s instructions, for all the analyses. Sampling was accom-plished in an air-conditioned room (22 ± 1 °C) to guarantee a stable temperature. After the equilibration time, the fiber was exposed to the headspace for 50 min at room temperature. Once sampling was finished, the fiber was withdrawn into the needle and transferred to the injection port of the GC-MS system. All the SPME sampling and desorption conditions were identical for all the samples. Furthermore, blanks were performed before each first SPME extraction and randomly repeated during each series. Quantitative comparisons of
tive peak areas were performed between the same chemicals in the different samples.
GC-Electron Impact Mass Spectrometry (EIMS) analyses were performed with a Varian (Palo Alto, CA) CP3800 gas chro-matograph equipped with a DB-5 capillary column (30 m × 0.25 mm × 0.25 μm; Agilent, Santa Clara, CA, USA) and a Varian Saturn 2000 ion trap mass detector. Analytical con-ditions were as follows: injector and transfer line temperatures were 220 and 240 °C, respectively; the oven temperature was programmed from 60 to 240 °C at 3 °C min−1; the carrier gas was helium at 1 mL min−1; splitless injection. The identifi-cation of the constituents was based on a comparison of the retention times with those of authentic standards, which com-prise all the compounds indicated in Table 3, except iso-pentyl acetate, valerolactone, 2-pentyl furan, (E,Z)-3,5-octadien-2-one, (E,E)-3,5-octadien-2-one, 1-nonen-3-ol, β-selinene and dihy-droactinidiolide, which were identified according to their MS spectra and linear retention indices (LRI), and on computer matching against commercial and home-made library mass spectra and data, specifically NIST 2000 and ADAMS 2007.16
Cytotoxicity assays
General. Each sample (∼1 g of freeze-dried powder) was extracted by stirring with 40 mL of methanol at 25 °C for 1 h and filtered through Whatman no. 4 filter paper. The residue was then extracted with an additional 40 mL portion of metha-nol. The combined methanolic extracts were evaporated under reduced pressure (rotary evaporator Büchi R-210, Flawil, Switzerland), re-dissolved in water at a concentration of 8 mg mL−1, and stored at 4 °C until determination of GI50 values (concentration that inhibited 50% of the net cell growth; expressed in µg mL−1). Ellipticine was used as a positive control.
Evaluation of cytotoxicity in human tumor cell lines. Four human tumor cell lines were used: HCT15 (colon carcinoma), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heat-inactivated FBS and 2 mM glutamine (MCF-7 and HCT15) or in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U per mL penicillin and 100 mg per mL streptomycin (HeLa and HepG2 cells), at 37 °C, in a humidi-fied air incubator containing 5% CO2. Each cell line was plated at an appropriate density (7.5 × 103 cells per well for MCF-7 and HCT15 or 1.0 × 104cells per well for HeLa and HepG2) in 96-well plates and allowed to attach for 24 h. Cells were then treated for 48 h with various extract concentrations. Following this incubation period, the adherent cells were fixed by adding cold 10% trichloroacetic acid (TCA, 100 µL) and incubated for 60 min at 4 °C. Plates were then washed with deionized water and dried; sulforhodamine B solution (0.1% in 1% acetic acid, 100 µL) was then added to each plate well and incubated for 30 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid. Plates were air-dried, the bound SRB was solubilized with 10 mM Tris (200 µL) and the
absor-bance was measured at 540 nm using an ELX800 Microplate Reader (Bio-Tek Instruments, Inc.; Winooski, VT, USA).17
Evaluation of cytotoxicity in a porcine liver primary cell culture. A cell culture was prepared from a freshly harvested porcine liver obtained from a local slaughter house, and it was designed as PLP2. Briefly, the liver tissues were rinsed in Hank’s balanced salt solution containing 100 U per mL peni-cillin, 100 µg per mL streptomycin and divided into 1 × 1 mm3 explants. Some of these explants were placed in 25 cm2tissue flasks in DMEM medium supplemented with 10% fetal bovine serum, 2 mM non-essential amino acids and 100 U per mL penicillin, 100 mg per mL streptomycin and incubated at 37 °C under a humidified atmosphere containing 5% CO2. The medium was changed every two days. Cultivation of the cells was continued with direct monitoring every two to three days using a phase contrast microscope. Before confluence was reached, cells were subcultured and plated in 96-well plates at a density of 1.0 × 104 cells per well, and cultivated in DMEM medium with 10% FBS, 100 U per mL penicillin and 100 µg per mL streptomycin.17
Evaluation of the antimicrobial activity
General. The methanolic extracts were re-dissolved in a 5% solution of DMSO in distilled water at 100 mg mL−1. Succes-sive dilutions were made from the stock solution and sub-jected to antibacterial and antifungal assays. Bacterial and fungal organisms were obtained from the Mycological Labora-tory, Department of Plant Physiology, Institute for Biological Research “Sinisa Stanković”, University of Belgrade, Serbia. DMSO (5%) was used as a negative control.
Antibacterial activity. The following Gram-positive bacteria: Staphylococcus aureus (ATCC 6538), Bacillus cereus (clinical isolate), Micrococcus flavus (ATCC10240), and Listeria monocyto-genes (NCTC7973) and Gram-negative bacteria: Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), Salmo-nella typhimurium (ATCC 13311), and Enterobacter cloacae (ATCC 35030) were used.
The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations were determined by the microdilution method. Briefly, a fresh overnight culture of bacteria was adjusted using a spectrophotometer to a concentration of 1 × 105 CFU mL−1. The requested CFU mL−1 corresponded to a bacterial suspension determined using a spectrophotometer at 625 nm (OD625). Dilutions of inocula were cultured on a solid medium to verify the absence of contamination and check the validity of the inoculum. Different solvent dilutions of the methanolic extract/fractions were placed in the wells contain-ing 100μL of tryptic soy broth (TSB) and afterwards 10 μL of inoculum was added. The microplates were incubated for 24 h at 37 °C.
The minimum inhibitory concentration (MIC) of each extract was detected following the addition of 40μL of iodo-nitrotetrazolium chloride (INT) (0.2 mg mL−1) and incubation at 37 °C for 30 min. The lowest concentration that produced a significant inhibition (around 50%) of the growth of the
teria in comparison with the positive control was identified as the MIC. The minimum bactericidal concentration (MBC) was determined by serial subculture of 10μL into microplates con-taining 100μL of TSB. The lowest concentration not showing growth after this subculturing was read as the MBC. Standard drugs, namely, streptomycin and ampicillin, were used as posi-tive controls. DMSO (5%) was used as a negaposi-tive control.
Antifungal activity. For the antifungal bioassays, the follow-ing microfungi were used: Aspergillus fumigatus (ATCC1022), Aspergillus ochraceus (ATCC12066), Aspergillus versicolor (ATCC11730), Aspergillus niger (ATCC6275), Penicillium funiculo-sum (ATCC 36839), Penicillium ochrochloron (ATCC9112), Peni-cillium verrucosum var. cyclopium (food isolate), and Trichoderma viride (IAM 5061). The micromycetes were main-tained on malt agar (MA) and the cultures were stored at 4 °C and subcultured once a month.18 The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile saline (≈1.0 × 103μL−1per well). The inocula were stored at 4 °C for further use. Dilutions of the inocula were cultured on solid MA to verify the absence of con-tamination and to check the validity of the inoculum.
MIC determination was performed by a serial dilution tech-nique using 96-well microtitre plates. The extracts were dis-solved in a 5% solution of DMSO and added to a broth malt medium with fungal inoculum. The microplates were incu-bated for 72 h at 28 °C. The lowest concentrations without visible growth (at the binocular microscope) were defined as MIC. The minimum fungicidal concentration (MFC) was deter-mined by serial subculture of 2μL in microtitre plates contain-ing 100 μL of malt broth per well and further incubation for 72 h at 28 °C. The lowest concentration with no visible growth was defined as the MFC, indicating 99.5% killing of the orig-inal inoculum. Bifonazole and ketoconazole were used as posi-tive controls.19
Statistical analysis. For all the experiments three samples (n = 3) were analyzed and all the assays were carried out in
triplicate. The results are expressed as mean values ± standard deviation (SD), except for antimicrobial assays. All stati-stical tests were performed at a 5% significance level using IBM SPSS Statistics for Windows, version 22.0. (IBM Corp., USA).
The differences between the flowering stages were analyzed using one-way analysis of variance (ANOVA). The fulfilment of the one-way ANOVA requirements, specifically the normal dis-tribution of the residuals and the homogeneity of variance, was tested by means of the Shapiro Wilk and the Levene tests, respectively. All dependent variables were compared using Tukey’s honestly significant difference (HSD) or Tamhane’s T2 multiple comparison tests, when homoscedasticity was veri-fied or not, respectively.
Principal component analysis (PCA) was applied as a pattern recognition unsupervised classification method. The number of dimensions to keep for data analysis was assessed by the respective eigenvalues (which should be greater than one), by Cronbach’s alpha parameter (that must be positive) and also by the total percentage of variance (that should be as high as possible) explained by the number of components selected. The number of plotted dimensions was chosen in order to allow meaningful interpretations.
Results and discussion
Proximate composition and dietary fiber
The results for the proximate composition value are shown in Table 1. At the first two flowering stages, water was the major component (more than 80 g per 100 g fw), but it drastically decreases at the post-flowering stage (19.6 g per 100 g fw). On a dry weight (dw) basis, carbohydrates were the most abundant macronutrients (more than 80 g per 100 g dw) at all flowering stages, showing slightly higher values at the full flowering stage. Most (around 70%) of these carbohydrates were present as total dietary fiber (TDF), especially in insoluble forms. In
Table 1 Nutritional and dietaryfiber composition (g per 100 g dw; mean ± SD, n = 9) of the three flowering stages of Opuntia microdasys (Lehm.)
F1 F2 F3
p-Value
Homoscedasticitya 1-Way ANOVAb Moisture (g per 100 g fw) 82.3 ± 0.2 b 86.5 ± 0.3 a 19.6 ± 0.2 c 0.363 <0.001 Fat (g per 100 g dw) 2.0 ± 0.2 b 1.6 ± 0.1 c 2.2 ± 0.1 a <0.001 <0.001 Proteins (g per 100 g dw) 6.3 ± 0.1 a 6.0 ± 0.1 c 6.2 ± 0.1 b 0.003 <0.001 Ash (g per 100 g dw) 9.6 ± 0.1 a 9.4 ± 0.1 b 9.1 ± 0.1 c <0.001 <0.001 Total carbohydrates (g per 100 g dw) 82.1 ± 0.3 c 83.1 ± 0.1 a 82.5 ± 0.1 b <0.001 <0.001
IDF (g per 100 g dw) 44 ± 1 a 42 ± 1 b 44 ± 1 a 0.181 <0.001
SDF (g per 100 g dw) 20 ± 1 a 16 ± 1 b 14 ± 1 c 0.166 <0.001
TDF (g per 100 g dw) 64 ± 1 a 58 ± 1 b 58 ± 1 b 0.909 <0.001
Energy (kcal per 100 g dw) 243 ± 2 c 254 ± 2 b 259 ± 2 a 0.912 <0.001 dw: dry weight; fw: fresh weight; F1: vegetative stage, F2: full flowering stage, F3: post-flowering stage. IDF: insoluble dietary fiber; SDF: soluble dietary fiber; TDF: total dietary fiber.aHomoscedasticity among the flowering stages was tested by the Levene test: homoscedasticity, p > 0.05;
heteroscedasticity, p < 0.05.bp < 0.05 indicates that the mean value of the evaluated parameter of at least one flowering stage differs from the others (in this case multiple comparison tests were performed). For each stage, means within a row with different letters differ significantly (p < 0.05).
comparison with other species of the genus Opuntia, the TDF values were higher than those reported by Ammar et al.20for O. ficus-indica and O. stricta. Furthermore, it is recommended that a third of TDF of the diet (approximately 20 g in the present case) should be soluble dietary fiber, and the distri-bution of dietary fiber in the three flowering stages is in agree-ment with the nutritional recommendations.21In fact, these results are noticeable, even when compared to the dietary fiber contents detected in some cereal sources,22raising the possi-bility of using O. microdasys flowers as a potential source of dietary fiber and/or to be added as a food ingredient to other food products to improve the fiber intake of the population.
In addition, O. microdasys flowers showed significant ash contents (higher than 9 g per 100 g dw) and proteins (higher than 6 g per 100 g dw), both components with minor di ffer-ences among the flowering stages. Fat was the least abundant component (around 2 g per 100 g dw), with slightly higher values at the post flowering stage. In general, the moisture content in F1 and F2 was similar to the one quantified in the cladodes (92 g per 100 g fw) and pulps (87 g per 100 g fw) of this species. Fat levels in all flowering stages were close to the ones detected in the pulp (2.46 g per 100 g dw), while protein contents were comparable to those measured in the cladodes (4.25 g per 100 g dw). Ash contents were lower than the ones quantified in the cladodes (16.4 g per 100 g dw) and pulps (16.4 g per 100 g dw), whilst carbohydrates were nearly the same.23The flowers’ nutritional profiles resulted in energetic values close to 250 kcal per 100 g dw, with the highest values being detected at the full flowering stage. These values are similar to those reported for O. ficus-indica and O. stricta flowers.20
Mineral composition
The content of mineral elements is one of the most important aspects influencing the use of edible flowers in human nutri-tion.24The mineral profile was composed of 4 major elements (K, Ca, Mg and Na) and 4 trace elements (Fe, Mn, Zn and Cu),
as shown in Table 2. Indeed, the mineral composition for O. microdasys during flowering stages shows that K was the pre-dominant component followed by Ca > Mg > Na > Fe > Mn > Zn > Cu. Except for the iron content, some significant di ffer-ences were found among the assayed flowering stages ( prob-ably due to the modifications related to the ripening of flowers), but the quantities of each mineral element maintain their relative proportions throughout these stages.
The low sodium content (1.2–1.5 mg per 100 g dw) is cer-tainly noteworthy, considering the deleterious effect of this mineral element for cardiovascular diseases.25 On the other hand, potassium (the most abundant element in O. microdasys) is one of the most important intracellular ions and is essential for the homeostatic balance of body fluids, besides controlling muscle contraction, particularly of the myocardium.26The consumption of food products rich in pot-assium is also recommended for the prevention of oncogenic diseases.27 Moreover, calcium, the second major element, is well-known for being one of the essential minerals needed for building the bones and teeth in animals and humans.
Despite the lower concentrations of microelements, when compared to those reported in other Opuntia species,20 the detected elements are also relevant: Cu (0.008–0.016 mg per 100 g dw), Mn (0.15–0.21 mg per 100 g dw) and Fe (0.19–0.21 mg per 100 g dw) play an important role in redox processes, besides acting as cofactors of different enzymes;28Zn (0.05–0.10 mg per 100 g dw) is, for instance, recognized as an essential element against prostate pain.29 Accordingly, O. microdasys flowers showed high potential as alternative sources of these mineral elements (despite the low bioavailability that characterizes some of the identified minerals) and might have applications as food supplements in meals and drinks.
Aroma volatiles of flower at three stages of maturity
Table 3 lists the volatile compounds identified in each of the flowering stages of O. microdasys. A total of 53 volatile com-pounds were detected (29 in F1, 30, in F2 and 28 in F3), but
Table 2 Micro and macroelements (mean ± SD, n = 9) of the three flowering stages of Opuntia microdasys (Lehm.)
F1 F2 F3
p-Value
Homoscedasticitya 1-Way ANOVAb Macroelements (mg per 100 g dw) Ca 21 ± 3 a 17 ± 2 b 23 ± 2 a 0.595 <0.001 Mg 13 ± 1 a 12 ± 1 b 10 ± 1 c 0.157 <0.001 Na 1.3 ± 0.1 ab 1.2 ± 0.1 b 1.5 ± 0.3 a <0.001 0.025 K 2909 ± 68 b 3711 ± 229 a 2564 ± 521 b <0.001 <0.001 Microelements (mg per 100 g dw) Cu 0.016 ± 0.001 a 0.013 ± 0.002 b 0.008 ± 0.001 c 0.141 <0.001 Fe 0.21 ± 0.01 0.19 ± 0.03 0.20 ± 0.02 0.001 0.385 Mn 0.21 ± 0.02 a 0.15 ± 0.01 b 0.15 ± 0.01 b 0.002 <0.001 Zn 0.06 ± 0.01 b 0.10 ± 0.01 a 0.05 ± 0.01 c 0.002 <0.001
dw: dry weight; fw: fresh weight; F1: vegetative stage, F2: full flowering stage, F3: post-flowering stage.aHomoscedasticity among the flowering
stages was tested by the Levene test: homoscedasticity, p > 0.05; heteroscedasticity, p < 0.05.bp < 0.05 indicates that the mean value of the evaluated parameter of at least one flowering stage differs from the others (in this case multiple comparison tests were performed). For each stage, means within a row with different letters differ significantly (p < 0.05).
only 12 were common to all the three stages. The identified compounds accounted for 92.3–96.5% of the total aroma. In general, each volatile compound is characterized by an odor threshold (varying from a few ppb to several ppm), so even if
the qualitative composition of different fruits is almost the same, the aroma may vary when the relative proportions are different30or even when minor constituents with a low odor threshold are present.31
Table 3 Comparative percentages of compounds in the volatile oils of the threeflowering stages of Opuntia microdasys (Lehm.)
Compound LRI F1 F2 F3
p-Value
Homoscedasticitya 1-Way ANOVAb
Hexanal 803 0.27 ± 0.05 nd nd 0.003 — Furfural 833 nd nd 0.42 ± 0.05 <0.001 — Furfuryl alcohol 856 nd 0.18 ± 0.05 nd <0.001 — 1-Hexanol 870 2.2 ± 0.1 b 0.8 ± 0.1 c 3.2 ± 0.2 a 0.029 <0.001 iso-Pentyl acetate 878 nd 0.62 ± 0.05 nd <0.001 — Ethyl pentanoate 900 0.60 ± 0.05 a 0.22 ± 0.04 c 0.40 ± 0.05 b 0.166 <0.001 Methyl hexanoate 929 5.6 ± 0.4 a 2.8 ± 0.1 c 3.1 ± 0.1 b 0.001 <0.001 Valerolactone 952 0.18 ± 0.03 b 0.20 ± 0.05 b 0.68 ± 0.05 a 0.371 <0.001 Benzaldehyde 963 nd nd 0.48 ± 0.05 <0.001 — 1-Heptanol 971 0.29 ± 0.04 b 0.15 ± 0.03 c 0.39 ± 0.05 a 0.671 <0.001 Hexanoic acid 979 0.40 ± 0.05 b 0.19 ± 0.05 c 0.82 ± 0.05 a 0.980 <0.001 6-Methyl-5-hepten-2-one 897 nd nd 0.54 ± 0.04 0.001 — 2-Pentyl furan 993 nd 0.61 ± 0.05 nd <0.001 — Ethyl hexanoate 998 4.8 ± 0.3 b 1.9 ± 0.1 c 6.1 ± 0.3 a 0.003 <0.001 1-Hexyl acetate 1010 nd 0.40 ± 0.04 0.61 ± 0.05 0.001 <0.001 Methyl heptanoate 1028 0.7 ± 0.1 nd 0.5 ± 0.1 <0.001 <0.001 p-Cymene 1028 nd 0.40 ± 0.04 nd <0.001 — Limonene 1032 2.2 ± 0.2 b 7.0 ± 0.3 a 2.3 ± 0.2 b 0.724 <0.001 iso-Octanol 1058 0.52 ± 0.05 nd nd 0.002 — γ-Terpinene 1063 nd 0.9 ± 0.1 nd <0.001 — (E)-2-Octenal 1063 nd nd 0.50 ± 0.05 <0.001 — (E,Z)-3,5-Octadien-2-one 1070 nd nd 1.6 ± 0.1 <0.001 — 1-Octanol 1072 0.7 ± 0.1 nd nd <0.001 —
cis-Linalool oxide (Furanoid) 1076 nd 0.40 ± 0.05 nd <0.001 —
(E,E)-3,5-Octadien-2-one 1079 nd nd 0.9 ± 0.1 <0.001 — 1-Nonen-3-ol 1080 0.21 ± 0.03 nd nd <0.001 — o-Guaiacol 1088 0.36 ± 0.05 nd 0.40 ± 0.05 0.001 <0.001 Ethyl heptanoate 1097 4.1 ± 02 nd nd <0.001 — Linalool 1101 nd 7.0 ± 0.4 2.7 ± 0.2 <0.001 <0.001 Nonanal 1104 nd nd 5.6 ± 0.3 <0.001 — Phenylethyl alcohol 1111 1.0 ± 0.1 nd nd 0.001 — Methyl octanoate 1128 2.1 ± 0.2 nd nd <0.001 — Camphor 1146 40 ± 1 c 48 ± 1 a 46 ± 1 b 0.605 <0.001 (E)-2-Nonenal 1164 nd nd 0.62 ± 0.05 <0.001 — 1-Nonanol 1172 0.9 ± 0.1 b 0.7 ± 0.1 c 1.1 ± 0.1 a 0.067 <0.001 4-Terpineol 1179 0.30 ± 0.05 nd nd <0.001 — 2-Decanone 1194 nd nd 0.9 ± 0.1 <0.001 — cis-Dihydrocarvone 1194 0.70 ± 0.05 nd nd <0.001 — Ethyl octanoate 1197 5.5 ± 0.4 nd nd <0.001 — n-Dodecane 1200 nd 2.1 ± 0.1 2.3 ± 0.1 0.002 <0.001 Methyl nonanoate 1227 4.0 ± 0.3 a 0.7 ± 0.1 c 2.5 ± 0.1 b 0.014 <0.001 Cumin aldehyde 1241 nd 0.56 ± 0.05 nd <0.001 — Carvone 1244 3.5 ± 0.3 b 15.8 ± 0.3 a 3.1 ± 0.2 c 0.522 <0.001 2-Undecanone 1293 nd 0.5 ± 0.1 1.6 ± 0.1 <0.001 <0.001 Ethyl nonanoate 1298 9.1 ± 0.4 a 2.1 ± 0.1 c 5.1 ± 0.4 b 0.054 <0.001 n-Tridecane 1300 nd 1.2 ± 0.1 nd <0.001 — 1-Nonyl acetate 1313 nd 0.21 ± 0.04 nd <0.001 — α-Copaene 1377 0.37 ± 0.05 0.10 ± 0.01 nd <0.001 <0.001 Ethyl decanoate 1397 0.28 ± 0.05 0.20 ± 0.03 nd <0.001 <0.001 β-Caryophyllene 1419 nd 0.18 ± 0.03 nd <0.001 — Aromadendrene 1440 0.41 ± 0.04 nd nd <0.001 — β-Selinene 1486 nd 0.10 ± 0.01 nd <0.001 — Dihydroactiniodiolide 1530 0.47 ± 0.05 nd nd <0.001 —
Percentage of identified compounds 94.8% 96.5% 92.3% —
F1: vegetative stage, F2: full flowering stage, F3: post-flowering stage; LRI: linear retention indices.aHomoscedasticity among the flowering
stages was tested by the Levene test: homoscedasticity, p > 0.05; heteroscedasticity, p < 0.05.bp < 0.05 indicates that the mean value of the evaluated parameter of at least one flowering stage differs from the others (in this case multiple comparison tests were performed). For each stage, means within a row with different letters differ significantly (p < 0.05).
In general, oxygenated monoterpenes were found to be the most important groups of volatiles during the maturation of flower, probably being the main contributors to the aroma in O. microdasys flowers. These compounds are widely used as fragrances and flavors in the cosmetic, perfume, drug and food industries. Another important group of volatiles was rep-resented by ester compounds, particularly methyl and ethyl esters of hexanoic, heptanoic, octanoic and nonanoic acids. Among the alcohols, 1-hexanol, known for conferring a fresh-mowed grass scent, was the main compound, whilst nonanal (which provides a soapy-fruity flavor) was the aldehyde detected in highest amounts, despite being only detected at the post flowering stage.32
The major compound at all flowering stages was camphor (40% in F1, 48% in F2 and 46% in F3), followed by ethyl non-anoate (9.1%) in F1, carvone (15.8% in F2) and ethyl hexnon-anoate (6.1%) in F3. Camphor (2-bornanone) derives naturally from the bark of the Cinnamomum camphora tree, but is also a major essential oil constituent in aromatic plants, such as the Greek sage (Salvia fruticosa), Spanish sage (Salvia lavandulifo-lia), Lavender cotton (Santolina insularis), and sweet worm-wood (Artemisia annua).33Camphor is known for its biological properties and industrial applications, despite being limited to a threshold value of 11% in medical products.34,35
When compared to other Opuntia species, the volatile com-position of O. microdasys is quite dissimilar, specifically due to the absence of camphor. In O. ficus-indica, for instance, 1-hexanol and germacrene D were the major volatile com-pounds.10These differences might be understood as an indi-cator of the high species-specificity of these types of compounds.
Cytotoxic activity
Results regarding the effects of the three flowering stages on four human tumor cell lines (MCF-7, HCT-15, HeLa and HepG2) are presented in Table 4. None of the tested extracts showed an inhibitory effect against the MCF7 cell line. In contrast, they were effective against all the remaining tumor cell lines. O. microdasys flowers were particularly active against the HCT15 cell line; among the tested flowering stages, the vegetative stage (F1) was the most effective in all the tested cell lines, as indicated by its lower GI50 values (97–185 μg mL−1).
Despite its cytotoxic activity on human tumor cell lines, the extracts of O. microdasys flowers did not show any hepato-toxicity in normal cells (PLP2), since the maximum assayed concentration (400 μg mL−1) had no significant inhibitory effect.
Antimicrobial activity
Results of the antibacterial activity towards pathogenic bac-teria (evaluated by the microdilution method), are presented in Table 5. The three flowering stages exhibited significant levels of antibacterial activity, but the full flowering stage (F2) turned out to be the most effective antibacterial agent, either considering its bacteriostatic (MIC varying from 0.312 mg
mL−1to 1.25 mg mL−1) or its bactericidal (MBC varying from 0.625 to 2.50 mg mL−1) effects. Regarding bacterial sensitivity, Staphylococcus aureus was the most susceptible species, whilst Listeria monocytogenes and Enterobacter cloacae stood out as the species with the highest resistance against the O. microdasys flower extracts. Comparing the results with those of the standard drugs ampicillin and streptomycin, F2 exhibi-ted higher activity than ampicillin against Pseudomonas aerugi-nosa and Salmonella typhimurium.
On the other hand, the extracts obtained from the vegeta-tive stage (F1) were the most acvegeta-tive in inhibiting the fungal growth (MIC varying from 0.95 mg mL−1 to 5 mg mL−1) and exerting fungicidal activity (MFC varying from 1.25 mg mL−1to 10 mg mL−1) (Table 5), somehow reflecting the compositional differences highlighted in Tables 2 and 3. Aspergillus versicolor and Penicillium funiculosum were the most susceptible fungal species, whereas Penicillium ochrochloron showed the highest resistance against the O. microdasys extracts. The tested stan-dards (bifonazole and ketoconazole) had lower MIC and MFC for all bacterial species.
The detected antimicrobial activity may be provided by the phenolic compounds present in the methanolic extracts. In fact, phenolic compounds may interact with the microor-ganism’s cell membrane or cell wall through hydrogen bonds involving their hydroxyl groups, thereby causing changes in membrane permeability and cell destruction.36,37 In fact, active natural compounds have been compared with repre-sentative antibacterial active ingredients commonly employed in medicine (e.g., chlorhexidine and Triclosan), to determine their effectiveness.38Considering some pre-set cri-teria from the relevant literature, agents with MIC values of isolated phytochemicals below 20 mg mL−1 may be con-sidered useful for therapeutic applications,39which classifies O. microdasys flowers as potential sources of compounds for these uses.
Table 4 Cytotoxic activity GI50 (μg mL−1) of methanolic extracts
obtained from the threeflowering stages of Opuntia microdasys (Lehm.)
Cell
lines F1 F2 F3
p-Value
Homoscedasti-citya 1-WayANOVAb
In human tumor cell lines
MCF7 >400 >400 >400 — — HCT15 97 ± 1 c 185 ± 1 a 126 ± 8 b <0.001 <0.001 HeLa 117 ± 4 c 232 ± 1 a 129 ± 2 b <0.001 <0.001 HepG2 238 ± 5 c 350 ± 5 a 278 ± 5 b 0.665 <0.001 In non-tumor cells PLP2 >400 >400 >400 — —
F1: vegetative stage, F2: full flowering stage, F3: post-flowering stage.
aHomoscedasticity among the flowering stages was tested by the
Levene test: homoscedasticity, p > 0.05; heteroscedasticity, p < 0.05.bp
< 0.05 indicates that the mean value of the evaluated parameter of at least one flowering stage differs from the others (in this case multiple comparison tests were performed). For each stage, means within a row with different letters differ significantly (p < 0.05).
Principal component analysis (PCA)
In the former section, the differences induced by the flowering stage were compared considering each parameter individually. As explained, several significant differences were found, but the parameter levels which best characterize each flowering stage could not be determined. Accordingly, in the present section, the results were evaluated considering data for all parameters (except for antimicrobial activity indicators) simul-taneously, by applying principal component analysis (PCA).
The plot of object scores and component loadings (Fig. 1) indicated that the first two dimensions (first: Cronbach’s α, 0.987; eigenvalue, 37.166; second: Cronbach’s α, 0.978; eigen-value, 28.570) account for most of the variance (90.0%) of all quantified variables (50.9% and 39.1%, respectively). Groups corresponding to each flowering stage (F1, F2 and F3) were completely individualized (objects corresponding to each stage were highlighted to facilitate the visualization), and the biplot also allows concluding which of the assayed parameters charac-terize better the assayed flowering stages. The vegetative stage (F1) is clearly typified by low levels of n-dodecane, camphor and
1-hexyl acetate, while presenting high quantities of fiber, manganese, methyl hexanoate, 1-octanol, hexanal and iso-octanol. Likewise, the full flowering stage (F2) is mainly charac-terized by low levels of fat, calcium, 1-hexanol, 1-heptanol, 1-nonanol, hexanoic acid and ethyl hexanoate, whereas it pre-sents high contents of potassium, zinc, furfuryl alcohol, iso-pentyl acetate, 2-iso-pentylfurane and p-cymene. Finally, the post-flowering stage (F3) stood out for having low quantities of water, ash, SDF, copper, magnesium, ethyl decanoate andα-copaene, simultaneously presenting raised levels of hexanoic acid, (E)-2-octenal, furfural, benzaldehyde, (E,Z)-3,5-octadien-2-one, (E,E)-3,5-octadien-2-one, valerolactone and 6-methyl-5-hepten-2-one.
Conclusion
The O. microdasys flowers in different ripening stages showed statistically significant differences in the proximate, mineral and volatile compound profiles, observed also in cytotoxic and antimicrobial properties. Even so, some overall
con-Table 5 Antimicrobial activity (MIC, MBC and MFC in mg mL−1) of the threeflowering stages of Opuntia microdasys (Lehm.)
Species
F1 F2 F3 Streptomycin Ampicillin
MIC MIC MIC MIC MIC
MBC MBC MBC MBC MBC Bacteria Staphylococcus aureus 0.450 0.450 0.312 0.04 0.25 0.625 0.625 0.625 0.10 0.40 Bacillus cereus 0.312 0.95 0.450 0.10 0.25 0.625 1.25 0.625 0.20 0.40 Micrococcus flavus 1.25 1.25 1.25 0.20 0.25 2.50 2.50 2.50 0.30 0.40 Listeria monocytogenes 3.75 1.25 2.50 0.20 0.40 5.00 2.50 5.00 0.30 0.50 Pseudomonas aeruginosa 0.95 0.450 0.95 0.20 0.75 1.25 0.625 1.25 0.30 1.20 Escherichia coli 0.95 0.95 0.95 0.20 0.40 1.25 1.25 1.25 0.30 0.50 Enterobacter cloacae 3.75 1.25 2.50 0.20 0.25 5.00 2.50 5.00 0.30 0.50 Salmonella typhimurium 0.625 0.312 0.625 0.25 0.40 2.50 0.625 2.50 0.50 0.75 Fungi Aspergillus fumigatus 2.50 5.00 1.25 0.15 0.20 5.00 10.0 5.00 0.20 0.50 Aspergillus versicolor 0.95 1.25 0.95 0.10 0.20 1.25 2.50 2.50 0.20 0.50 Aspergillus ochraceus 1.85 10.0 5.00 0.15 1.50 2.50 12.5 10.0 0.20 2.00 Aspergillus niger 1.25 10.0 2.50 0.15 0.20 5.00 12.5 10.0 0.20 0.50 Trichoderma viride 2.50 5.00 2.50 0.15 1.00 5.00 10.0 5.00 0.20 1.00 Penicillium funiculosum 0.95 1.25 1.25 0.20 0.20 1.25 2.50 2.50 0.25 0.50 Penicillium ochrochloron 5.00 5.00 5.00 0.20 2.50 10.0 10.0 10.0 0.25 3.50 Penicillium verrucosum 5.00 5.00 3.75 0.10 0.20 10.0 10.0 5.00 0.20 0.30
F1: vegetative stage, F2: full flowering stage, F3: post-flowering stage; MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration; MFC: minimum fungicidal concentration.
clusions might be drawn: O. microdasys presented high con-tents of dietary fiber, potassium and camphor; regarding the bioactivity, the performances against HCT15 cells, Staphylo-coccus aureus, Aspergillus versicolor and Penicillium funiculo-sum deserve special attention. The vegetative stage (F2) showed the highest levels of cytotoxicity and antifungal
activity, whilst the full flowering stage (F3) gave the best results for antibacterial activity. By analysing all the results simultaneously through principal component analysis, it was possible to characterize the chemical and bioactive profiles, which better characterize each of the flowering stages, while identifying their most distinctive features. These results
Fig. 1 Plots of object scores (flowering stages) and component loadings (selected parameters). Object scores were highlighted for a better visualization.
could be useful to select the optimum flowering stage for a determined application.
Competing interests
The authors declare no competing financial interests.
Acknowledgements
The authors are grateful to Fundação para a Ciência e a Tecno-logia (FCT, Portugal) for financial support to CIMO (strategic project PEst-OE/AGR/UI0690/2011) and the ALIMNOVA research group (UCM-GR35/10A). J.C.M. Barreira and R.C. Cal-helha thank FCT, POPH-QREN and FSE for their grants (SFRH/ BPD/72802/2010 and SFRH/BPD/68344/2010, respectively). The Serbian Ministry of Education is also acknowledged for the Science and Technological Development Grant No. 173032.
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